Alarming multidrug resistance in Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis: Antibiotic resistance patterns and occurrence of selected resistance genes

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.


Introduction
Mastitis is an inflammatory condition of the mammary gland and udder tissue in cows, resulting in abnormal and reduced milk yield.Typically, mastitis develops as a result of the immune system's response to microorganisms invading the udder through the teat canal.It can also be caused due to physical damage, exposure to harmful chemicals, or high temperatures affecting the udder.It is one of the most common dairy sector diseases [1].Mastitis may be subclinical or clinical, and can be either environmental or contagious [2].Clinical mastitis (CM) cases include one or more of the following symptoms: irregular milk production, inflamed udder, clumps and clots in milk and systemic symptoms, including fever, fatigue, loss of appetite and depression, while the symptoms of sub-clinical mastitis (SCM) include low production of milk, and poor milk quality however the udder appears normal [3].During SCM, in order to combat invasive pathogens, white blood cells (WBCs) such as macrophages and neutrophils move from bloodstream to the udder tissues.This results in an increased somatic cell count (SCC), and as SCC level rises, milk production declines.Milk's protein content changes dramatically over time.Casein, the main milk protein decreases, while proteins of low nutritional value increase, negatively impacting milk [4].Reduced milk production and discarding milk from unhealthy animals result in financial losses.Other factors contributing to losses include removing milk that contains antibiotic residues, early cow removal and replacement with expensive ones, veterinarian fees, prescription costs, extra hourly payments, and decreased commercial worth of removed cows [5].
Many contagious pathogens are responsible for mammary gland infection.Udder and teat skin of the cow are common habitats for contagious pathogens, which are commonly disseminated during the milking process from one cow to another.SCM is mostly caused by a highly contagious bacterium Staphylococcus aureus developing resistance and producing different virulence elements, such as endotoxins and other dangerous proteins.Mastitis in cows causes major economic losses and is associated mainly with S. aureus [1].S. aureus is mostly found on or in the udder of cows, commonly transferred from one teat to another within a single cow or between cows while milking [6].
S. aureus contains multiple virulence factors that contribute to its toxicity.Adhesins, superantigens, capsules, and toxins are some of these factors.Adhesins are protein or glycoproteins on the bacterium's surface that allow it to adhere to host cells.Toxins are chemicals produced by bacteria that are capable of damaging the host cells and tissues.Superantigens are poisons that can activate a high number of immune cells, resulting in an overly aggressive immunological response.Capsules are coverings that protect the bacterium from the host's immune system [7,8].A variety of infections can be caused by S. aureus, including pneumonia, bone and joint infections, skin and soft tissue infections, and bloodstream infections.The way diseases develop varies depending on the type of infection.For example, in skin and soft tissue infections, bacteria might get into the body through a wound or cut, giving rise to the infection.In cases of bloodstream infections, the bacteria have the ability to enter the bloodstream and travel to other body parts, resulting in sepsis [9,10].
In staphylococci, antibiotics primarily target nucleic acids, the cell envelope, and ribosomes to stop bacterial growth.Recent targeted drug development programs have identified many new targets, including the cell division machinery's FtsZ and the protease ClpP [11].The utilization of vast amounts of antibiotics for both humans and farm animals led to the S. aureus emergence, which displayed resistance to a variety of drugs.Reports show that the low-and middle-income countries are more actively contributing to the rise in antibiotic resistance rates compared to high-income countries [12,13].Resistance can either develop from mutations in genetic elements or via the horizontal resistance gene transfer encoded by movable genetic components like plasmids, staphylococcal cassette chromosomes, and jumping genes.In S. aureus, antibiotic resistance genes include the most prevalent tetracycline resistance genes (tetL, tetO, tetM, tetK) and erythromycin resistance genes (ermA, ermC, ermB).Three commonly recognized mechanisms confer tetracycline resistance to organisms: (i) Efflux by tetracycline-specific pumps, (ii) Ribosomal protection, and (iii) Enzymatic inactivation.Erythromycin resistance can arise through different mechanisms, such as efflux pumps, modification of target sites, and alteration of ribosomes [11].S. aureus may develop multidrug resistance in one of two ways.First, multiple genes that code for drug resistance can accumulate over time within the bacteria; this accumulation often occurs on resistance plasmids.Second, increased expression of the genes that make multidrug efflux pumps that release a number of drugs may contribute to multidrug resistance [14].
S. aureus is a crucial pathogen for both humans and animals.When treating staphylococcal infections, resistance to drugs is a significant problem [15].S. aureus exhibits significant resistance to certain antimicrobial classes, which restricts choices for therapy available to veterinary and human medicine [16].The dairy sector and the food safety of humans are greatly affected by the multi-drug resistant (MDR) strains [17].Multidrug resistance has been increased all over the world that is considered a public health threat.Several recent investigations reported the emergence of MDR bacterial pathogens from different origins that increase the necessity of the proper use of antibiotics.Additionally, antimicrobial susceptibility testing is routinely applied to identify the antibiotic of choice and screen for emerging MDR strains [12].The presence of methicillin-resistant S. aureus (MRSA) in unpasteurized milk poses a major threat to public health [18].Public health concerns have recently increased with the appearance of extremely virulent community acquired-MRSA strains [19].The world health organization has named nosocomial infections caused by MRSA among the world's three most challenging infectious illnesses due to its severity and prevalence [20].The emergence of MRSA is attributed to the mecA resistance gene, and the presence of the tetK gene is recognized as a contributing factor to the development of Tetracycline resistance [14].Gentamicin, tobramycin, and kanamycin resistance are all conferred by the aacA-aphD gene, which encodes an aminoglycoside modifying enzyme and blaZ gene causes resistance to beta-lactam antibiotics, such as amoxicillin and ampicillin [21].
Staphylococcus aureus exhibits an outstanding variety of virulence features, allowing it to survive in extreme host conditions and result in tissue colonization, tissue damage, and lifethreatening systemic infections.The Panton-Valentine leukocidin (PVL) toxin is the main virulence factors of S. aureus, that targets WBCs.PVL enters the host plasma membrane and creates a pore or hole, it has a strong affinity for leukocytes, whereas other toxins, such as leukocidin and γ-hemolysin, are cytotoxic to WBCs and red blood cells, respectively [7].In the context of cattle, S. aureus can induce mastitis by entering the mammary gland via the teat canal and colonizing the gland, resulting in the release of enzymes and toxins that kill host cells and tissues [22].
To ensure effective decision-making regarding the treatment of animals with antibiotics and to evade the emergence of antibiotic resistance, the pattern of S. aureus's antibiotic resistance in raw milk needs to be monitored [23].Therefore, the current study was designed to find out the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis.

Ethical statement
This study was approved by the Advanced Studies and Research Board (ASRB) (Dir/A&R/ AWKUM/2022/9396) of the Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan, Pakistan.The oral permissions were obtained from the farm managers regarding milk and blood collection.

Study design
Milk samples were obtained from 543 lactating cows, including Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from six different dairy farms across Pakistan.The samples were collected in sterilized bottles containing potassium dichromate (0.01 mg) as a preservative using aseptic techniques to avoid bacterial contamination and quickly transported in an icebox to CVS & AH AWKUM for further processing and examination of the milk contents.

Somatic cell count (SCC)
From the milk samples, microscopic slides were prepared and the SCC was assessed using the procedure of Sharma et al. [24].Two fields of 1 cm 2 were made on the clean glass slide.Ten microliters of milk were added to each field and then left to air dry.After drying, xylene was added for 2-3 minutes to defat the slides, which were then fixed by adding 95% ethanol for 5 minutes and left to air dry.After fixation, 10% Giemsa stain was added to each field for 30 minutes after that, rinse with tap water.The slides were all examined through a 100X oil immersion lens of the microscope.

Isolation and confirmation of S. aureus
S. aureus isolation and identification were performed using mannitol salt agar (MSA) plates (Oxoid Ltd., Hampshire, England) [25].The plates were incubated for 18 to 24 hours at 37˚C and were then observed for S. aureus colonies.For the isolation of pure colonies, the S. aureus colony was picked from the first culture and was sub-cultured on the same medium-MSA, then kept at 37˚C for 18 to 24 hours.Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+) [26].As a quality-control strain, S. aureus ATCC25923 was used.

Stock preparation
Following the manufacturer's instructions, Luria-Bertani (LB) broth (Sigma-Aldrich Merck, Germany) was made.Fresh S. aureus colonies were added to the LB broth, and then the mixture was incubated stationary in a bacteriological incubator for 24 hours at 37˚C.The broth's turbidity after incubation showed S. aureus growth.For glycerol stock preparation, 500 μL of LB broth containing S. aureus was added to 1000 μL of 70% glycerol (Bio-Rad Inc., USA) in eppendorf and refrigerated at -40˚C until further usage.

DNA extraction
For DNA extraction of all confirmed S. aureus isolates, Chelex1 100 resin (15-50 mesh) (Bio-Rad Inc., USA) was used [27].In a sterile falcon tube, a five percent solution of Chelex was prepared.After that, 70 μL of 5% Chelex solution was added into a sterile eppendorf through a pipette, and then a few pure colonies from a fresh culture of the bacteria were added into it via a sterile wire loop.Using a pipette, the Chelex and the bacterial colonies were mixed gently.After this, the mixture was heated in a water bath for 30 minutes at 45˚C, followed by centrifugation for 10 seconds at 5000 rpm.In the new eppendorf, the supernatant was collected.The DNA was confirmed by using an agarose gel (Bio-Rad Inc., USA) of 1%.

Molecular identification of S. aureus
All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene, which is unique to this bacterium [28].S. aureus reference strain ATCC25923 and molecular grade water were applied as respective positive and negative controls.The details of the primer (Bio-Rad Inc., USA) used is given in Table 1.For the Polymerase Chain Reaction (PCR), a total of 20 μL of reaction mixture were used [28].DNA was initially denatured for five minutes at 94˚C then 30 cycles were completed using the following thermal cycling protocol for PCR: second denaturation at 94˚C for 30 seconds; annealing at 52˚C for 30 seconds; extension at 72˚C for 30 seconds; and final amplification for five minutes at 72˚C [28].Finally, the PCR samples were subjected to electrophoresis through a two percent agarose gel (Bio-Rad Inc., USA).

Detection of antibiotic resistance genes through PCR
Antibiotic resistance genes, mecA, tetK, blaZ, and aacA-aphDwere detected and amplified using conventional PCR.From our lab, S. aureus strains positive for mecA, tetK, blaZ, and aacA-aphD genes as a positive control, and molecular grade water as a negative control was used [27].The details of the primers used are given in Table 1.For the PCR, a total of 20 μL of reaction mixture was used.DNA was initially denatured for 10 minutes at 95˚C, then 35 cycles were completed using the following thermal cycling protocol for PCR: second denaturation at 95˚C for 30 seconds; annealing at 56˚C, 63.6˚C, 55˚C, and 57˚C for 30 seconds for tetK, mecA, aacA-aphD, and blaZ respectively; and extension at 72˚C for 30 seconds.During the final amplification phase, at 72˚C, the complete single DNA strand was polymerized for 10 minutes [33,34].The desired genes were verified using an agarose gel of 1.5 percent.

Statistical analysis
Data was collected from well-known dairy farms and inserted into Excel sheets.Statistical analysis was done to determine relationships between different breeds and farms of cattle with the prevalence of S. aureus.P < 0.05 was considered significant when using the chi-square test.
Pearson's Correlation Coefficient was determined to measure the relationship between the identified resistance genes and the tested antibiotic classes.The SPSS version 22 was used to analyze the data.

Results
Mastitis is an infectious condition that affects the mammary gland and occurs as a result of injury with the aim of neutralizing or killing the infectious agents and preparing the path for healing and the restoration to normal function.Mastitis may be subclinical or clinical and depending on the major reservoir and transmission methods, can be either environmental or contagious.Mastitis may have a number of etiological causes; however, bacteria are most commonly to blame.S. aureus is among the most common bacteria causing mastitis.S. aureus is making the infection more and more challenging, because with the passage of time it evolves from being mono-drug resistant to multi-drug resistant e.g MRSA.In this study 543 bovine milk samples were collected from different dairy farms in Pakistan.Subclinical mastitis was determined in the samples by evaluating the somatic cell count.Then subclinical bovine mastitis associated S. aureus were isolated and studied.The prevalence of S. aureus in SCM was assessed, the isolates antibiotic susceptibility was checked and antibiotic resistance genes in these isolates were determined.

Detection of sub-clinical mastitis
Depending on the quantity of somatic cells in one ml of milk, the health status of cows was determined.The SCC was done for all 543 samples.Cows were considered to have SCM if the SCC exceeded 200,000 cells/ml in milk.Out of 543 total milk samples, 57.09% (n = 310) had subclinical mastitis, of which 104 were Red Sindhi, 100 were Sahiwal, 61 were Cholistani, and 45 were Holstein Friesian (Table 2).

Phenotypic characteristics of the recovered S. aureus isolates
The S. aureus on MSA showed strong growth with pen pointed yellow colonies, displaying mannitol fermentation.The colonies revealed a round and well-defined morphology, differentiating them from other bacteria on MSA.Gram staining of S. aureus revealed Gram-positive cocci.The retrieved S. aureus isolates were poisitive for catalase and coagulase tests.

Prevalence of S. aureus in SCM positive samples
A total of 310 milk samples positive for SCM were examined for S. aureus presence.S. aureus was detected in 30.32% (94/310) milk samples.S. aureus was confirmed by its phenotypic characteristics and was identified molecularly through the species-specific nuc gene.

Antibiotic susceptibility testing
AST was performed on all 94 isolates of S. aureus.According to the CLSI guidelines, all verified S. aureus isolates were divided into three categories: resistant, intermediate, and susceptible for all antibiotics tested.Out of 94 isolates, 88% exhibited resistance to at least one of the antibiotics, including Amoxicillin, Gentamycin, Amikacin, Tetracycline, Levofloxacin, Cefoxitin, Lincomycin and Sulfamethoxazole/Trimethoprim.The isolates of S. aureus exhibited the highest levels of Lincomycin resistance (84.04%), followed by Ampicillin (45.74%),Amoxicillin (42.5%), and Tetracycline (17.02%), while the least resistance was shown to Sulfamethoxazole/ Trimethoprim (3.19%) and Gentamycin (6.38%) (Table 4).

Detection of antibiotic resistance genes
Through PCR, mecA, tetK, aacA-aphD and blaZ resistance genes were checked in all S. aureus isolates.Out of 94 samples, the blaZ gene was discovered in 52 samples, indicating its relatively widespread presence.In comparison, tetK gene was found in 44 samples, showing a lower frequency than blaZ.The mecA gene appeared in 16 samples whereas, aacA-aphD was found in just 13 samples.The Pearson's Correlation Coefficient between the identified resistance genes and the tested antibiotic classes was estimated in the overall S. aureus isolates.A positive correlation (r = 0.54) was found between the presence of blaZ gene and Penicillins resistance, between the presence of tetK gene and Tetracyclines resistance (r = 0.75), and between the presence of mecA gene and Cephamycins resistance (r = 0.72).The correlation between the aacA-aphD gene and Aminoglycosides resistance was r = 0.32.

MDR S. aureus isolates
A total of 94 S. aureus isolates were tested for antibiotic resistance phenotypically, in which 47 isolates were determined to be MDR.Isolates were classified as MDR based on the criteria outlined by [35].Among these MDR isolates, 23.40% (11 samples) exhibited resistance to Cefoxitin, and hence were classified as MRSA.Notably, 97.87% MDR isolates were non-susceptible to Lincomycin, with significant rates of non-susceptibility observed for Ampicillin (74.46%),Amoxicillin (72.34%),Tetracycline (63.82%) and Amikacin (25.53%).In contrast, only 6.38% of MDR isolates were non-susceptible to Sulfamethoxazole/ Trimethoprim, while 14.89% each were non-susceptible to Gentamycin and Levofloxacin.As shown in Table 5, 91.48% of MDR S. aureus isolates carried the blaZ gene, while 72.34% had the tetK gene, 29.74% had the mecA gene, and 23.40% carried the aacA-aphD gene.The Pearson's Correlation Coefficient analysis in the MDR isolates reveals a positive correlation between the resistant phenotypes and the resistance genes.The correlation between the Penicillin's resistance and the presence of the blaZ gene was r = 0.69.Cefoxitin resistance was positively correlated (r = 0.73) with the presence of the mecA gene.Similarly, Tetracyclines resistance was positively correlated (r = 0.72) with the presence of tetK gene.The correlation between the Aminoglycosides resistance and the presence of aacA-aphD gene was r = 0.59.Breed-wisely, SCM was found to be most prevalent in Sahiwal (57.14%) and Red Sindhi (57.14%), followed by Cholistani (57%) and Holstein Friesian (56.96%).A research by Bachaya et al. [43], found that SCM is common in crossbred cows (36%) in Muzaffar Garh, Pakistan.Dar et al. [25] reported a prevalence of 74.61% and 25.38% of SCM in crossbred and native cows, respectively.
S. aureus was prevalent in this study at a rate of 30.32 percent (n = 94 / 310).Compared to this, the prevalence in other countries was recorded as 43.1%, 46.6%, 50.0%, and 30.6% in China, USA, Poland, and Ethiopia, respectively [39,[44][45][46].A higher occurrence of S. aureus in SCM milk samples was recorded by Ren et al. [47] from southern Xinjiang China which was 77.38%, while the prevalence of S. aureus in Beijing reported by Wang et al. [48] was 46.2%.On the other hand, from two different regions of Ethiopia, 42.85% and 51.2% prevalence rates of S. aureus were reported by Bude and Mengesha [3] and Abebe et al. [49], respectively.The S. aureus prevalence in the previous studies and current study can vary due to several reasons, such as study design, sampling method, population characteristics, and geographic location [50].
The bacteria become resistant to a drug by one of these mechanisms; inactivating or modifying the drug enzymatically, modifying the binding site of the drug, drug efflux, protecting the target by displacing the drug, circumvent mechanisms involving gaining of new drug resistant targets [11].In the current research, 47 (50%) of the 94 S. aureus isolates were MDR.A previous study from Pakistan reported nearly the same prevalence (44.44%) of MDR bacteria in the milk samples [36].A comparable MDR prevalence (46.8%) was observed in another study carried out in China [44].The current study's MDR prevalence was significantly greater than reports from other countries, such as Indonesia (10%), Greece (13.3%), and Kenya (29.67%) [51][52][53].The isolates of S. aureus exhibited the highest levels of Lincomycin resistance (84.04%), followed by Ampicillin (45.74%),Amoxicillin (42.5%), and Tetracycline (17.02%).This high resistance rate is because of MDR strains' emergence, escalated by the excessive misuse of antibiotics in veterinary practice.In contrast, the isolates showed the highest susceptibility to Sulfamethoxazole/ Trimethoprim (95.74%) and Gentamycin (89.36%).The isolates' susceptibility to Sulfamethoxazole/Trimethoprim is attributed to the synergistic effect of the drug, while Gentamycin is used less frequently, resulting in lower resistance towards it [54].The high prevalence of MDR S. aureus strains among SCM cases indicates the need for a more vigilant approach toward the use of antibiotics in veterinary practices.Additionally, it is advised to periodically test the antimicrobial sensitivity of the pathogens before usage and monitor the rational use of medications.
The mecA, tetK, aacA-aphD and blaZ resistance genes were checked in all S. aureus isolates.The blaZ gene was found in 52 (55%) samples, indicating its relatively high prevalence.Whereas, 44 (46.80%) of S. aureus isolates had the tetK gene, the mecA gene was found in 16 (17.02%)isolates, and aacA-aphD was found in just 13 (13.82)samples.The two main mechanisms S. aureus employs to develop β-lactam resistance are the penicillinase production and penicillin-binding protein modification [55].S. aureus becomes resistant to tetracycline by activating the efflux pump encoded by the tetK gene [19].Gentamicin and Amikacin belong to the aminoglycoside class of antibiotics, and transferrable plasmids that encode bifunctional aminoglycoside-modifying enzymes like aacA-aphD can cause resistance to them [56].Similarly, Amoxicillin and Ampicillin, which are both penicillin-class antibiotics, are not any more effective compared to other antibiotics against bacteria that make beta-lactamase enzymes such as blaZ [57].An earlier investigation from China carried out by Qu et al. [34] reported nearly the same occurrence of mecA (16%) and tetK gene (31%), whereas, a relatively higher frequency of aacA-aphD (23%) and blaZ gene (95%) in S. aureus isolates.Liu et al. [58] discovered that 12.90% of the S. aureus isolates carried the mecA gene in large-scale dairy farms in China.In contrast, Mbindyo et al. [52] from Kenya found a higher prevalence of mecA (25%) and a lower prevalence of tetK (0%) and blaZ gene (41%) in the S. aureus isolates.
In this study, there is discrepancy between the presence of antibiotic resistance genes and the frequency of resistance in the phenotype.It is common for bacteria to have an antibiotic resistance gene but not being resistant to the concerned drug.This is due to the fact that the presence of an antibiotic resistance gene does not guarantee that the bacteria will be resistant to the drug.Additional factors such as the presence of other genes, expression of the gene, and environmental conditions can all affect whether or not the bacteria will develop antibiotic resistance [59].
In the current research the raw milk is significantly contaminated with S. aureus including the MDR-MRSA strains that carried the antibiotic resistance genes.This resistance can be transmitted from cows to humans through the consumption of contaminated milk or contact with infected animals, directly endangering human health [6].Furthermore, the persistence of antibiotic-resistant bacteria in the agricultural setting, such as on dairy farms, can facilitate the spread of resistance genes throughout the wider ecosystem, worsening the global antibiotic resistance crisis.

Conclusions
It is concluded that the raw milk of the studied breeds of cows is significantly contaminated with antibiotic resistant S. aureus including the MDR isolates and MRSA strains.The isolated S. aureus harbored various resistance genes responsible for the absorbed phenotypic resistance.The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases present a serious risk to public health.Therefore, this study has provided valuable information that may be helpful to reduce public health risks associated with acquisition of MDR-MRSA along with the milk value chain.Consequently, we recommend continuous surveillance and monitoring of antibiotic resistant S. aureus, including the MDR-MRSA to reduce the emergence and spread of the milk-borne drug-resistant S. aureus strains, to protect the public health and promote sustainable one health is Pakistan.

Table 5 . Antibiotic resistance pattern of the MDR S. aureus isolate and the expressed resistance genes.
[42]his study, out of 543 total milk samples, 57.09% (n = 310) had SCC levels above 200,000, indicating SCM.In Pakistan, Najeeb et al.[36]reported a 39.2% SCM prevalence, 80.8% of the total cases were caused by S. aureus, and 18.8% of milk samples contained MRSA based on AST and 6.5% based on the molecular study.A review article authored by Bari et al.[37]discovered that SCM varied in cows, from 18% to 87% in India.Chen et al.[38]published a study in 2022 indicated that in seven regions of China, the estimated prevalence of subclinical mastitis ranged between 36.4% and 50.2%.Similar research was done by Tesfaye et al.[39]and found a comparatively higher prevalence of SCM, i.e., 69.4%.A previous study from Muzaffar Garh Pakistan, reported that 45% of crossbred cows had SCM[40].In South Ethiopia's Hawassa milk shed, dairy cattle herds were the subject of a study by Abebe et al.[41]they reported a 59.2% prevalence of SCM.Sefinew et al.[42]reported 9.7% and 37.2% prevalence of CM and SCM, respectively in milk collected from local zebu breeds and zebu-Holsteins cross breeds in Ethiopia.